WO2021211441A1 - Modified sugar polymers with low anticoagulant activity and therapeutic activity - Google Patents

Abstract

Embodiments relate to the synthesis and utilization of size-defined and artificial sulfated hepbiuronic acid (sHBA) polysaccharides with low anticoagulant activity. sHBA has the ability to bind, modulate and/or inhibit key proteins involved in the inflammation process including the complement system and a defense enzyme, myeloperoxidase.

Description

MODIFIED SUGAR POLYMERS WITH LOW ANTICOAGULANT ACTIVITY AND THERAPEUTIC ACTIVITY INCORPORATION BY REFERENCE TO ANY PRIORITY APPLICATIONS [0001] This application claims priority to U.S. Provisional Application No. 63/009,290 entitled “MODIFIED SUGAR POLYMERS WITH LOW ANTICOAGULANT ACTIVITY AND THERAPEUTIC ACTIVITY” filed on April 13, 2020, the entirety of which is hereby incorporated by reference. BACKGROUND [0002] Heparan sulfate (HS), including the well-known drug heparin, is a class of polysaccharides built on a [4-Glucuronic acid-1ȕ-4-N-acetyl-glucosamine-1Į-]_n ([4-GlcA-1β- 4-GlcNAc-1α-]_n) sugar backbone and plays essential roles in mammals. This backbone is modified by various enzymes including N-deacetylase, N- and O-sulfotransferases, and C₅- epimerase in the Golgi apparatus of the cell. The many structural variants of HS observed in humans have been hypothesized to differentially modulate signaling, proliferation, inflammation, and hemostasis during health and disease. A favored hypothesis is that the pattern of HS domains (comprised of both N-sulfo (NS) and N-acetyl (NA) domains) encodes information that modulates HS interaction with proteins like growth factors, cytokines, and clotting factors. [0003] Due to the nature of glycan biosynthesis and the variety of cells types and tissues as well as developmental changes, the HS class of molecules found in mammals is very heterogeneous. Purification of single molecular entities at large scale is virtually impossible due to the original heterogeneity of the animal-derived HS class of molecules as well as the inherent relatively non-selective nature of the existing chemical methods. In addition, the animal-derived HS class of molecules has lot-to-lot variability and an insecure supply chain (e.g., many suppliers pooling raw materials and unscrupulous spiking in over-sulfated chondroitin that caused many deaths in the 2008 contaminated heparin episode). Alternatively, current organic chemical methods to make synthetic HS (e.g., Arixtra, the synthetic pentasaccharide) are laborious, generate much toxic waste, typically only make small sugar chains, and often product-specific. [0004] Heparin has the most potent activity, effectively binding or signaling virtually all protein players in the “heparanome.” Thus, heparin has potential for many treatments, but is simply “too active” for safe and selective therapy. For example, it is known that heparin and some of its derivatives (namely 6-O-sulfated versions) are effective at reducing inflammation, but these polymers have potential for bleeding problems as well as possess lot-to-lot variability. [0005] Therefore, safe and effective treatments for inflammation are needed. Some current treatments use agents that bind to proteins of the complement system to treat inflammatory-related diseases. PMX-53 (PEPTECH, LTD.) is used to treat rheumatoid arthritis and psoriasis, and binds to the C5 complement protein. [0006] Novel structural variants of HS, which have more selective bioactivities, less off-target effects, and less potential for causing hemorrhage than ‘native’ HS and heparin, should be useful in the design of new therapeutics. SUMMARY [0007] Some embodiments of the present invention relate to a pharmaceutical composition comprising at least one sulfated hepbiuronic acid (sHBA) disaccharide unit. In some embodiments, the number of sulfated hepbiuronic acid (sHBA) disaccharide units is from 2 to 1,000. In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier. [0008] Some embodiments of the present invention relate to a method of reducing inflammation in a subject. The method comprises determining a subject in need of treatment for inflammation; and administering to the subject a therapeutically effective amount of the pharmaceutical composition, wherein the administration results in reduced inflammation in the subject. [0009] In some embodiments, the inflammation is inflammation derived from a microbial (viral or bacterial) infection. [0010] In some embodiments, the method further comprising administering another therapeutic agent to said subject. [0011] In some embodiments, the pharmaceutical composition and the other therapeutic agent are co-administered. [0012] In some embodiments, the other therapeutic agent is an interferon, a SARS- CoV-2 RNA-dependent RNA polymerase inhibitor, a cap-dependent endonuclease, a protease inhibitor, or a spike inhibitor. [0013] In some embodiments, both the pharmaceutical composition and the other therapeutic agent are dispersed or dissolved together in a pharmaceutically-acceptable carrier. [0014] In some embodiments, the administration is intravenous or subcutaneous administration. [0015] In some embodiments, the administration is pulmonary administration. [0016] In some embodiments, wherein the administration is of a solution comprising the pharmaceutical composition or pharmaceutically acceptable salt in a concentration between 0.1 mg/ml and 500 mg/ml. [0017] In some embodiments, the pharmaceutical composition or pharmaceutically acceptable salt thereof administered to the subject is in an amount from about 0.1 mg to about 1,000 mg of the pharmaceutical composition per kg of body weight of the subject. [0018] These and other features, aspects, and advantages of the present invention will become better understood with reference to the following description and appended claims. BRIEF DESCRIPTION OF THE DRAWINGS [0019] The drawings illustrate certain embodiments of the technology and are not limiting. For clarity and ease of illustration, the drawings are not made to scale and in some instances, various aspects may be shown exaggerated or enlarged to facilitate an understanding of particular embodiments. [0020] Fig. 1A depicts a mass spectrum of dye-derivatized non-sulfated HBA disaccharides, showing a main peak at a mass-to-charge ratio of 531. [0021] Fig. 1B depicts a mass spectrum of dye-derivatized mono-sulfated HBA (sHBA) disaccharides, showing a main peak at mass-to-charge ratio of 611. [0022] Fig. 2 depicts an Agarose/StainsAll gel analysis of HBA and various sulfated HBA polymers evidencing different levels of sulfation. Two natural polysaccharides, chondroitin sulfate (CS; shark) and heparin (Hp; porcine intestinal), and the synthetic hyaluronan standards (L; 100 kDa or 30 kDa for top versus bottom band, respectively) calibrate the gel. For these polymers, the color changes from blue when unsulfated to purple then yellow with increasing sulfation levels in general. [0023] Fig.3A depicts examples of the synthesis of hepbiuronic acid (HBA) using the PmHS enzyme (Step #1) and showing a gel of a sample at preparation made at the ~5 to 20 milligram scale. After sulfation (Step #2), the sHBA polymer migrates faster due to increased charge density (i.e. more anionic polymer chains). The gel results are from Agarose/StainsAll gels of HBA and sHBA polysaccharides. [0024] Fig. 3B depicts that two independent batches of a 35-kDa sulfated HBA (sHBA) polymer were shown to be homogeneous preparations (2 ^g yields tight dark bands) and seem similar in charge density. The gel results are from Agarose/StainsAll gels of HBA polysaccharides. The color changes from blue to purple with polymer sulfation. Three natural polysaccharides, chondroitin sulfate (CS), heparan sulfate (HS; porcine) and heparin (Hp) are also depicted. [0025] Fig. 4 depicts a human diagnostic coagulation assay comparing heparin to sHBA. [0026] Fig. 5 depicts an SDS-PAGE analysis of affinity bead experiments to identify human sHBA-binding targets. [0027] Fig. 6 shows a biocompatibility test where human lysosomal extracts were found to cleave the sHBA compound. [0028] Fig. 7 shows the NMR data for a sHBA compound according to one embodiment of the invention. [0029] Fig.8 is a bar chart showing IL-6 cytokine levels in mouse lung lavage fluid following infection with influenza A strain PR8 and treatment with sHBA in vivo. DETAILED DESCRIPTION [0030] In the Summary Section above and the Detailed Description Section, and the claims below, reference is made to particular features of the invention. It is to be understood that the disclosure of the invention in this specification includes all possible combinations of such particular features. For example, where a particular feature is disclosed in the context of a particular aspect or embodiment of the invention, or a particular claim, that feature can also be used, to the extent possible, in combination with and/or in the context of other particular aspects and embodiments of the invention, and in the invention generally. [0031] Embodiments of the invention relate to the synthesis and utilization of sulfonated or sulfated hepbiuronic acid (sHBA) polysaccharides. In some embodiments, the sHBA polysaccharides are size-defined and sulfation-specific. These sHBA molecules were found to have the ability to modulate and/or inhibit key proteins involved in the inflammation process, including the complement system and a defense enzyme, myeloperoxidase. [0032] Some embodiments described herein relate to a pharmaceutical composition comprising at least one sulfated hepbiuronic acid (sHBA) repeating disaccharide unit (O- sulfated [-4-GlcA-1-beta-4-Glc-1-alpha]). Some embodiments comprise formulations of polymers having 2 to 1,000 repeating disaccharide units of sHBA in a polymer. Other embodiments may comprise formulations of polymers having 5-500, 50-250, 100-150 polymers or any number in between these ranges. Some embodiments comprise formulations of polymers having 2 to 1,000 repeating disaccharide units of both unsulfated HBA and O- sulfated sHBA in a single polymer chain. In some embodiments, the glucose in the sHBA is less than 100% sulfated. In other embodiments, the glucose in the sHBA is 90%, 80%, 70%, 60% or less sulfated or any range of sulfation in between these values. [0033] Other embodiments described herein relate to a method of reducing inflammation in a subject by treatment with sHBA compositions or formulations. In one embodiment, a subject in need of treatment for inflammation is determined; and a therapeutically effective amount of the pharmaceutical composition comprising sHBA is administered to the subject, wherein the administration results in reduced inflammation in the subject. In other embodiments, the inflammation is derived from a viral infection, such as the SARS-CoV-19 virus or influenza. [0034] As used herein, a “therapeutically effective amount” refers to a sufficient amount of sHBA polymer to treat a subject in need of treatment for inflammation, at a reasonable benefit/risk ratio applicable to any medical treatment. It will be understood, however, that the total daily usage of sHBA will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular subject will depend upon a variety of factors including the disorder being treated and the severity of the disorder; the specific composition employed, the age, body weight, general health, sex and diet of the subject; the time of administration, route of administration, and rate of excretion of sHBA employed; the duration of the treatment; drugs used in combination or coincidental with sHBA; and like factors well known in the medical arts. For example, it is well known within the skill of the art to start doses of the compound at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. In addition, a “therapeutically effective amount” is the amount that will elicit the biological or medical response of a tissue, system, or subject that is being sought by a researcher or clinician, and in particular elicit some desired therapeutic or prophylactic effect as against a viral infection. [0035] One of skill in the art recognizes that an amount may be considered therapeutically “effective” even if the condition is not totally eradicated or prevented, but it or its symptoms and/or effects are improved or alleviated partially in the subject. [0036] In some embodiments, the composition comprises from about 5% to about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% by weight of sHBA polymer, and preferably from about 30% to about 90% by weight of sHBA polymer, based upon the total weight of the composition taken as 100% by weight. [0037] Other ingredients may be included in the pharmaceutical composition, such as other active agents, preservatives, buffering agents, salts, a pharmaceutically acceptable carrier, or other pharmaceutically-acceptable ingredients. [0038] In some embodiments, the method further comprises administering another therapeutic agent to the subject as combination therapy is known to be especially effective for infectious disease treatment (e.g., HIV virus in AIDS). In some embodiments, the therapeutic agents can be but not limited to SARS-CoV-2 RNA-dependent RNA polymerase inhibitor, a cap-dependent endonuclease, protease inhibitors, a spike inhibitor, or interferons. Examples of suitable RNA polymerase inhibitors include, but are not limited to, Remdesivir (development code GS-5734) by Gilead Sciences. In some embodiments, the other therapeutic agent is interferon, such as a pegylated interferon. Examples of suitable interferons include, but are not limited to, Pegylated interferon-alpha-2a (brand name PEGASYS®), Pegylated interferon-alpha-2b (brand name PEG-INTRON®), interferon alfacon-1 (brand name INFERGEN®), pegylated interferon lambda and/or a combination thereof. In some embodiments, the antiviral composition and the other therapeutic agent are co-administered. In one embodiment, the order of administering the antiviral compound and the other therapeutic agents can be in any order. [0039] In some embodiments, both the pharmaceutical composition and the other therapeutic agent are dispersed or dissolved together in a pharmaceutically-acceptable carrier. [0040] In some embodiments, the administration of the antiviral composition is of a solution comprising sHBA or pharmaceutically acceptable salt in a concentration between 0.1 mg/ml and 500 mg/ml. [0041] The dosage may range broadly, depending upon the desired effects and the therapeutic indication. The daily dosage regimen for an adult human patient may be, for example, an injected dose of between 0.01 mg and 3000 mg of sHBA, preferably between 1 mg and 700 mg, e.g. 5 to 200 mg, or between about 0.1 mg and about 1,000 mg of sHBA per kg of body weight of the subject. The dosage may be a single one or a series of two or more given in the course of one or more days, as is needed by the subject. In some embodiments, the compounds are administered for a period of continuous therapy, for example for a week or more, or for months or years. In some embodiments, sHBA, or a pharmaceutically acceptable salt thereof, can be administered less frequently compared to the frequency of administration of an agent within the standard of care. In some embodiments, sHBA, or a pharmaceutically acceptable salt thereof, can be administered one time per day. For example, sHBA, or a pharmaceutically acceptable salt thereof, can be administered one time per day to a subject suffering from a SARS-CoV-2 infection. In some embodiments, the total time of the treatment regime with sHBA, or a pharmaceutically acceptable salt thereof, can be less compared to the total time of the treatment regime with the standard of care. [0042] In instances where human dosages for sHBA have been established for at least some condition, those same dosages may be used, or dosages that are between about 0.1% and 500%, more preferably between about 25% and 250% of the established human dosage. Where no human dosage is established, as will be the case for newly-discovered pharmaceutical compositions, a suitable human dosage can be inferred from ED50 or ID50 values, or other appropriate values derived from in vitro or in vivo studies, as qualified by toxicity studies and efficacy studies in animals. [0043] In cases of administration of a pharmaceutically acceptable salt, dosages may be calculated as the free acid. As will be understood by those of skill in the art, in certain situations it may be necessary to administer the compounds disclosed herein in amounts that exceed, or even far exceed, the above-stated, preferred dosage range in order to effectively and aggressively treat particularly aggressive diseases or infections. [0044] Dosage amount and interval may be adjusted individually to provide plasma levels of the active moiety that are sufficient to maintain the modulating effects, or minimal effective concentration (MEC). Dosages necessary to achieve the MEC will depend on individual characteristics and route of administration. However, HPLC assays or bioassays can be used to determine plasma concentrations. Dosage intervals can also be determined using MEC value. Compositions should be administered using a regimen that maintains plasma levels above the MEC for 10-90% of the time, preferably between 30-90% and most preferably between 50-90%. In cases of local administration or selective uptake, the effective local concentration of the drug may not be related to plasma concentration. [0045] It should be noted that the attending physician would know how to and when to terminate, interrupt, or adjust administration due to toxicity or organ dysfunctions. Conversely, the attending physician would also know to adjust treatment to higher levels if the clinical response were not adequate (precluding toxicity). The magnitude of an administrated dose in the management of the disorder of interest will vary with the severity of the condition to be treated and to the route of administration. The severity of the condition may, for example, be evaluated, in part, by standard prognostic evaluation methods. Further, the dose and perhaps dose frequency, will also vary according to the age, body weight, and response of the individual patient. [0046] Pharmaceutical compositions disclosed herein can be evaluated for efficacy and toxicity using known methods. For example, the toxicology of a particular pharmaceutical composition may be established by determining in vitro toxicity towards a cell line, such as a mammalian, and preferably human cell line. The results of such studies are often predictive of toxicity in animals, such as mammals, or more specifically, humans. Alternatively, the toxicity of particular pharmaceutical composition in an animal model, such as mice, rats, rabbits, or monkeys, may be determined using known methods. The efficacy of a particular pharmaceutical composition may be established using several recognized methods, such as in vitro methods, animal models, or human clinical trials. When selecting a model to determine efficacy, the skilled artisan can be guided by the state of the art to choose an appropriate model, dose, route of administration and/or regime. [0047] The disclosed embodiments are suitable for various routes of administration, depending upon the particular carrier and other ingredients used. For example, the pharmaceutical compositions can be injected intramuscularly, subcutaneously, intradermally, or intravenously. In some embodiments, the administration is of a solution comprising sHBA or pharmaceutically acceptable salt in a concentration between 1 mg/ml and 500 mg/ml. The pharmaceutical composition can also be administered via mucosa, such as intranasally or orally. Another delivery path for the pharmaceutical composition is the pulmonary route where the drug is dispersed into the lungs via a nebulized solution or an inhalable powder. [0048] A class of polymers based on a hepbiuronic acid (HBA, [4-Glucuronic acid- 1β-4-Glucose-1α-]_n) backbone are extremely similar to heparosan, the unmodified backbone of both heparin and heparan sulfate. The chemical structures for the repeating hepbiuronic acid (HBA) backbone structure (bottom) compared to the natural heparosan backbone (top) are shown below. The arrows highlight the structural differences of a hydroxyl in HBA versus a N-acetyl group in heparosan.

[0049] Sulfation, by either chemical or enzymatic means, of the HBA backbone produces sulfated HBA (sHBA). The artificial sugar polymer sHBA can be manufactured by chemoenzymatic polysaccharide synthesis followed by sulfation, where the selectivity of enzyme catalysts was found to vastly improve the homogeneity of resulting products. The more controlled, uniform manufacture process also removes animal sourcing and its resultant lot-to- lot variation. As shown below, this artificial sugar polymer sHBA was found to bind some, but not all, of the proteins that were known to interact with HS or heparin. [0050] In one embodiment, a chemoenzymatic method of synthesizing sHBA was used to make the HBA backbone and then O-sulfation was used to add the various levels of modification for protein binding. In one embodiment, a PmHS (Pasteurella multocida heparosan synthase) enzyme was used to make the HBA backbone, but the invention is not limited to wild-type sequence PmHS enzymes; chimeric and mutated enzymes may be substituted as well as orthologs from other genera (e.g., E. coli, Avibacter). Chemoenzymatic synthesis of the HBA backbone: [0051] An enzyme was used to polymerize the HBA backbone in vitro. This process involves preparing the recombinant PmHS-G10 (a chimeric PmHS1/PmHS2 synthase) in E. coli, reacting the UDP-sugar donors (UDP-Glc and UDP-GlcA) with the enzyme in a reaction buffer (pH 4-8 with divalent cation such as Mg²⁺ or Mn²⁺), and isolating the polysaccharide product ([-4-GlcA-1-beta-4-Glc-1-alpha]n). The chain size or molecular weight (MW) of the polymer may be altered by either of two proven routes: (a) adding a primer (e.g., a short [di-, tri-, tetrasaccharide] heparosan oligosaccharide, a glucuronide glycoside, etc) at the correct ratio to donor or (b) by multiple UDP-sugar additions over time. Each method has its own benefits; the former yields a tight size distribution due to reaction synchronization and has the potential for adding a chemical functionality (e.g., fluorescent probe, biotin, etc) at the reducing terminus of the chain. For example, by controlling the primer (or acceptor) to donor ratio, 9.8, 14.5, or 27.3 kDa HBA polymers (with more primer forcing the smaller chain sizes) were prepared. [0052] However, it may be more convenient to make larger MW polymers using an alternate approach where the HBA is made in de novo polymerizations without a primer. In the initiation step, one UDP-sugar in the enzyme’s donor site is added to another UDP-sugar that is sitting in the PmHS enzyme’s acceptor pocket. While this reaction is slower to start (i.e. no acceptor to prime the reaction), it does not require an extra starting material. The de novo method can be used to make large MW polymers. In one example, we synthesized a 70 kDa HBA polymer in one such preparation. [0053] The HBA polymers may be purified by a combination of standard biochemical methods including solvent extraction (e.g., chloroform), alcohol precipitation (e.g., 2.5 volumes of ethanol), ion exchange (e.g., Q Sepharose) or reverse phase (e.g, C18) chromatography, and/or ultrafiltration (Amicon filtration units). [0054] After the HBA polymers are prepared, sulfation reactions are used to form sHBA. In one example, a low temperature, aqueous O-sulfation method may be used to prepare sHBA types (O-sulfated [-4-GlcA-1-beta-4-Glc-1-alpha]n). The sulfation level of the HBA is controlled by using various ratios of the sulfation reagent (e.g., sulfur trioxide complexes) to polysaccharide and/or the reaction conditions (e.g., time, temperature). Basic (high pH, e.g., pH>9) aqueous reaction conditions appear to be self-limiting such that over-sulfation of the sugar at the many hydroxyl groups on each sugar ring does not occur. In another example, chlorosulfonic acid in anhydrous formamide, an anhydrous organic solvent, can yield higher levels of O-sulfation of HBA, with more than one sulfate per monosaccharide (i.e. sulfates added to different sugar ring positions preferentially). Recombinant HS O-sulfotransferase- based enzymatic sulfation is an additional method to the chemical routes as this enzyme does modify HBA. As described in the Examples below, it was discovered that radiochemical incorporation of ³⁵S sulfate was found using this approach to sulfonate HBA. [0055] After the polymerization and sulfation reactions (aqueous or organic sulfation conditions), the sHBA intermediates may be further purified by anion exchange chromatography with linear salt gradients (0.1 to 1 M) on Q Sepharose resin (GE), reverse- phase extraction, and/or ultrafiltration against water to concentrate and de-salt the target- containing fractions. [0056] Some sulfated HBA polysaccharides (sHBA) with relatively low anticoagulant activity compared to heparin were made. The activity was measured by the human diagnostic aPPT assay in human plasma samples. In other experiments, various polymers were immobilized onto a solid phase bead and then employed in an affinity-based approach to identify binding proteins in human plasma. After extensive washing, proteins that were found to be tightly bound to the sHBA were eluted with high salt concentration. It was discovered that members of the complement system and myeloperoxidase were found bound to sHBA in the highest levels, as explained more fully below in the Examples. [0057] It should be realized that other methods of making HBA and sHBA polymers of varying sizes are also contemplated. For example, de novo synthesis may be used to make polymers of 20-100 kDa average. For example, polymers of approximately 28 kDa and 70 kDa were made using de novo synthesis methods. In addition, aqueous sulfated HBA of 28 kDa and 70 kDa were also made by the methods described herein. For various small oligosaccharides (i.e. less that ~20 monosaccharide units per chain), two methods are useful: (a) step-wise addition of single sugars to a growing HBA chain or (b) by controlling the stoichiometry of the primer to UDP-sugars. It should be realized that HBA/sHBA polymers of any size are contemplated by the methods described herein. [0058] In initial biocompatibility tests, it was observed that sHBA was degradable by enzymes of the human lysosome. Thus, sHBA administration to mammals as part of a treatment regimen should not be susceptible to having unwanted tissue accumulation as has been seen with other artificial products. [0059] Formulations that include the sHBA molecule may be useful to treat a variety of diseases having inflammatory components. For example, sHBA may be useful for treatment of Covid-19, the disease derived from the SARS-CoV-2 virus, a type of coronavirus. In this example, a long-term goal is to help preserve the lungs from the body’s over-reaction to the coronavirus, thereby patients should be able to breathe for a long time to come. This treatment should also be suitable for treating diseases in which other microbes (e.g., influenza, SARS) or sepsis (i.e. bacteria in the blood) trigger the innate immune system resulting in a “cytokine storm”, rampant inflammation, mis-targeted membrane attack complexes, and white cell attacks. Likewise, other health conditions with dysregulated inflammation that should be amenable to sHBA include, but is not limited to, autoimmune diseases, Alzheimers, asthma, traumatic brain injury, and ischemia, cardiovascular/blood vessel problems. [0060] Sulfated HBA polysaccharides (sHBA) with low anticoagulant activity compared to heparin were used in an affinity-based approach to identify binding proteins in human plasma. These binding proteins include members of the complement system and myeloperoxidase. Both types of proteins are known or strongly suspected to trigger and/or involved in inflammation and tissue damage (especially of blood vessels & lungs) that is spawned by many diseases including microbial infection (virus and bacteria), auto-immunity (e.g., lupus), cardiovascular problems, asthma, and neurological problems (e.g., Alzheimers. Parkinsons). Major factors in this complement-induced damage is the activation of a 'cytokine storm' and the white blood cell attack of the patient's own tissue; this “over-reaction” by the patient's defense system often causes morbidity beyond the disease itself. Myeloperoxidase, while normally employed to kill invaders or damaged cells, if over-active or mislocalized, can damage tissues with its hypochlorous acid product. [0061] sHBA is a biodegradable glycan mimicking the natural heparin/HS system, thus distinct from most other classes of current complement-active drugs in structural class (thus potential for synergism in combination therapies), with multiple modes of action and predicted enhanced biocompatibility. Example 1: Synthesis and uses of anti-inflammatory sugar-based polymers. Methods & Results [0062] 1. Preparation & Analysis of various sHBA polymers. [0063] First, HBA polysaccharide (ref: Lane RS, St Ange K, Zolghadr B, Liu X, Schäffer C, Linhardt RJ, DeAngelis PL. Expanding glycosaminoglycan chemical space: towards the creation of sulfated analogs, novel polymers and chimeric constructs. Glycobiology.2017 Jul 1;27(7):646-656) (the starting material shown in the lane 0S in part 1b below) was prepared by chemoenzymatic synthesis from UDP-GlcA and UDP-Glc in the presence of a PmHS-based (Pasteurella multocida heparosan synthase) catalyst in the presence of a divalent cation (e.g., Mn²⁺, Mg²⁺ at 0.1-20 mM) at neutral pH (e.g., pH ~6-8 with ~20-50 mM Tris or Hepes buffer) for 1-4 days at ~10-40qC. [0064] After purification of the HBA backbone, a chemical sulfation reagent was added to the HBA (general conditions: sulfur trioxide-trimethylamine complex, ~0.7-2 M aqueous NaOH, 0-20ºC, ~1-24 hours) to create O-sulfated-Glc units (~50% or ~100% level for lanes aS vs aS’ in part 1b, respectively) as determined by disaccharide analysis (part 1a below). In an alternative synthesis, multi-sulfated HBA (mS) was prepared by an organic solvent sulfation method (general conditions: chlorosulfonic acid in neat anhydrous formamide, 0-6ºC, 1-16 hrs, then quenched by excess water addition). In general for sHBA (sulfated HBA) purification, a combination of ethanol precipitation (2.5-3 volumes, 0.2-0,5 NaCl), ultrafiltration (3-kDa MWCO) and/or anion exchange (e.g., Q Sepharose with NaCl elution) were used to purify the materials before use. [0065] 1a) LC-MS of disaccharide analysis: An AMAC dye-tagged disaccharide method was used to analyze the parental and sulfated HBA polymers. In general, the samples were digested with an enzyme to disaccharides, tagged with a fluorescent hydrophobic dye (AMAC), separated via reverse phase HPLC then sprayed into a tandem mass spectrometer. The parental, unsulfated HBA disaccharide had the expected mass (531 Da) based on its structure. The sulfated HBA had the mass addition corresponding to a single sulfate addition to a single hydroxyl per disaccharide, resulting in a final mass of 611 Da. [0066] Detailed Method: HBA and derivatives were digested by recombinant heparinase III (10 mU) in 100 μL of digestion buffer (50 mM NH₄Ac, 2 mM CaCl₂, pH 7.0) at 37°C overnight. The reaction was terminated by eliminating the enzyme by passing through 3-kDa molecular weight cut-off spin columns (Millipore; Billerica, MA). The filter unit was washed twice with 200 μL distilled water and the combined filtrate was lyophilized. The dried samples were AMAC-labeled by adding 10 μL of 0.1 M AMAC in DMSO/acetic acid (17:3, v/v), incubating at room temperature for 10 min, followed by adding 10 μL of 1 M aqueous sodium cyanoborohydride, and incubating for 1 h at 45°C. A mixture containing two kinds of disaccharide standards (HS 0S standard, Iduron, Manchester, UK) prepared at 12.5 ng/μL was similarly AMAC-labeled and used for each run as an external standard. The resulting samples were centrifuged at 12,000 × g for 10 min. Finally, supernatant was collected and stored in a light resistant container at room temperature until analyzed. [0067] LC-MS analyses were performed on an Agilent 1200 LC/MSD Instrument (Agilent Technologies, Inc., Wilmington, DE) equipped with a 6300 ion-trap, a binary pump and a Poroshell 120 EC-C18 column (3 × 50 mm, 2.7 μm; Agilent). Mobile phase A was 50 mM NH₄Ac in water and mobile phase B was methanol. A gradient of 10–35% B for 10 min was run with a flow rate of 250 μL/min at 45°C. The electrospray was set in negative ionization mode with a skimmer potential of -40.0 V, a capillary exit of í40.0 V and a source temperature of 350°C. Mass range of the spectrum was 300–900 m/z. Nitrogen (8 L/min, 40 psi) was used as drying and nebulizing gas. As shown in Fig. 1A, non-sulfated HBA disaccharides were found to elute as a main peak with a mass-to-charge ration of 531, with a smaller peak at 531 (due to natural carbon, hydrogen isotopic abundances). As shown in Fig. 1B, mono-sulfated HBA (sHBA) disaccharides were found to have a higher mass-to-charge ratio of 611, with smaller peaks at 612 and 613. [0068] 1b) Electrophoretic Analysis of HBA and sHBA. Agarose gels (1X Tris- acetate-EDTA TAE, 1-2%, 30-80V, 0.5-4 hrs) were used to separate the polymers according to size and charge density. The gels were then stained with StainsAll (Sigma) dye that both detects the band as well as gives a charge density output based on color. The modified materials run faster on gels due to higher charge density than the parental HBA and also exhibit a color shift in this stain system (Fig. 2). [0069] This agarose gel (1.5%) depicts HBA with various sulfation levels (starting HBA, 0S; sulfated HBA, aS, aS’, mS). In addition to the expected mobility shift, Stains-All dye can discern the sulfation state of the polymer as a color shift from blue to purple to yellow as the sulfation level increases. For reference, the natural GAGs (shark chondroitin sulfate C, CS; porcine heparin, Hp, with 1 or 2 sulfates/disaccharide, respectively) show the same color- staining pattern. L, hyaluronic acid standards (Hyalose LoLadder, 100 kDa & 30 kDa shown). [0070] Examples of creating sHBA at different levels of sulfation at the ~5 to 20 milligram scale are shown in Figs. 3A and 3B. Two batches of a prototype 35-kDa sulfated HBA (sHBA) polymer are shown to be homogeneous preparations (2 μg yields tight dark bands) and seem similar in charge density (Fig.3B). As shown in Fig 3B, the heparin and HS polymers have a much wider size distribution (1 μg yields weaker, wider bands), which was confirmed by the light scattering analyses (polydispersity index M_w/M_n: sHBA = 1.01-1.02 versus 1.3 = heparin or 1.7 = HS; for reference, ‘1’ is an ideal perfect polymer). [0071] 2. Coagulation assay comparing heparin to sHBA. The sHBA (~100% mono-sulfated) was tested in the activated partial thromboplastin time (APTT) assay because it is used to monitor the effect of heparin dosage on clotting in the clinic. The human diagnostic assay on an automated analyzer (Diagnostica Stago STA Compact Coagulometer; Veterans Administration Hospital; Oklahoma City, OK) was performed with the standard clinical protocol with the normal, healthy pooled human control plasma as the sample for testing. The effect of the addition of sHBA or the heparin standard on the time to clot was compared. Briefly, each sample-plasma mixture (100 μL) was added to 100 μL of APTT reagent (Diagnostica Stago PTT reagent). After 4 min incubation at 37°C, 100μL of CaCl2 (30 mM) is added and the time to clot formation determined. All controls (normal and abnormal heparin controls) were within the established statistical values. [0072] Fig. 4 shows that the sHBA has much less coagulation potential than the drug heparin as seen in this human diagnostic assay. This indicates that sHBA, when administered to a patient, should result in fewer bleeding issues as compared to heparain. [0073] 3. HBA was enzymatically sulfated with 6-O-sulfotransferases. As an alternative sulfation method, recombinant human O-sulfotransferase (a Golgi enzyme that makes heparin in the body) was tested in a radiochemical assay with HBA. Radioactive ³⁵S from the PAPS donor was incorporated (9.8K disintegrations per min [dpm] versus the negative control 150 dpm in a [³⁵S]PAPS incorporation radioassay); this sulfation method is an alternative to the chemical methods described in part 1. [0074] 4. SDS-PAGE analysis of affinity bead experiments to identify human sHBA-binding targets. As detailed below, human plasma was interrogated with immobilized sHBA polysaccharides. Human plasma from healthy adults was incubated with various beads with immobilized (i) heparin drug, (ii) sHBA (~100% mono-sulfated), or (iii) no sugar (as a negative control) overnight with mixing by rolling at 37ºC. The beads were then extensively washed (~2-3 hours) with PBS buffer, and eluted with a small volume of 0.5 M NaCl, then by a second step with 1.5 M NaCl. The eluted fractions were concentrated & desalted by ultrafiltration then subjected to SDS-PAGE analysis with Coomassie Blue staining. Fig. 5 shows that heparin binds many proteins (lanes 3 and 4), and sHBA binds a smaller number of species (lanes 6 and 7) indicating its relative selectivity. Here, comparing lane 6 to lanes 3 and 4, one of the smaller MW (~60% of the way down this Coomassie Blue-stained SDS-PAGE gel, predicted ~36 kDa) proteins actually seems to prefer sHBA over heparin. [0075] 5. Proteomic identification of sHBA-binding proteins. These sHBA- binding proteins were analyzed via peptide mapping/mass spectrometry (Table 1). The eluted proteins from sHBA-affinity beads (see Fig. 5, lane 6) were subjected to a standard “direct injection proteomics” protocol (i.e., trypsin digestion and analysis by electrospray mass spectroscopy (Thermos Orbitrap Lumos spectrometer), and the peptides were compared to a database of all known human proteins by a matching program (Univ. of Oklahoma HSC Core laboratory) whereby the various peptides were identified by their masses dictated by sequence and known human post-translational modifications. The peptides with the highest signals are listed in Table 1.

[0076] Some proteins are likely from background binding of the very highest abundance plasma proteins to beads (e.g., serum albumin, transferrins not removed completely by wash steps, marked with *), but some of the most prevalent proteins isolated using the sHBA beads are the components the complement system & the enzyme myeloperoxidase (marked with ***) which appear to have high affinity for the sHBA based on their high signals. Two of the most abundant species detected were key components of the complement system: C1q, part of the initial protein complex of the classical pathway, and C3, a central player where all three complement pathways converge. [0077] Both sHBA-binding proteins are pivotal parts of the innate immune system. C3 is the most abundant complement protein in plasma, but C1q is found at rather low abundance.⁴⁴ The other players in the complement pathways (e.g., C2, C4, C5) that are at similar or higher plasma abundances than C1q were not detected, suggesting that the C3 and C1q proteins possess a high affinity for sHBA. The beads without polysaccharide (‘mock’ lanes in Fig.6) did not bind these 2 species, but bound the most abundant plasma proteins such as albumin and lactotransferrin in a non-specific fashion. The C1q and C3 proteins are also not part of the “CRAPome” database, the repository of ‘sticky’ proteins known to bind to various naked bead matrices. [0078] Bioassay tests to validate the complement lead detected in the affinity chromatography screening were performed. Commercial ELISA-based colorimetric-output assay used to diagnose patients with complement system genetic defects or acquired disease (#COMPL300RUO; Svar Inc., Sweden) were tested. Microplates with three different ‘triggers’ immobilized in separate wells were used as independent probes of the classical, lectin, or alternative pathways. It was found that all the pathways were inhibited by sHBA and heparin to various degrees, confirming that sHBA was indeed ‘complement-active’ (Table 2). In a sheep blood hemolysis assay to look for the classical pathway, it was discovered that sHBA also inhibited the destruction of the red cells. The anticoagulant activity of various sHBA preparations was very low in comparison to heparan sulfate and heparin as measured by a colorimetric enzyme assay used to measure the quantity of heparin in human plasma (Coamatic Heparin Kit; Diapharma, West Chester, OH). Comparing the complement inhibition and the anticoagulant datasets (the ‘anti-inflammatory / anticoagulant ratio’ or AI/AC in Table 2) allowed a rough estimate that sHBA may have a ~20- to 300-fold safety margin in treating complement dysregulation compared to unfractionated heparin, depending on the complement pathway. This may be an important factor underlying the selectivity of sHBA as compared to heparin as a treatment for a patient’s hyper-inflammatory state without resulting in the patient having undesirable bleeding problems following treatment.

^{^} Table.^2:^Inhibitory^Effects^of^Various^Heparinoids^Relative^to^Heparin^ ^

CP= Svar Classical Pathway, MP= SVAR Lectin Pathway, AP= Svar Alternative Pathway Anticoag = Diapharma Assay % Relative to Heparin Activity is the percent when compared to equivalent molar concentration of heparin set as ‘100%.’ AI/AC = Anti-Inflammatory/Anticoag Ratio = ratio is the ratio of a complement inhibition activity in a specific bioassay divided by anti-coagulation activity. *= averaged single determination [0079] As mentioned above and shown in Table 2, the bioactivity of heparin (normalized to ‘1’ or ‘100%’; porcine intestine) was compared to two different preps of sHBA with high sulfation (sHBA #1 and #2 preparations from Fig. 3B; almost all mono-sulfated disaccharides), heparan sulfate (HS; porcine intestine), and a sHBA with very low sulfation levels (sHBALow: average ~27% mono-sulfated disaccharides) in a variety of bioassays. The AI/AC ratios suggest that sHBA could be safer than heparin and more effective than HS. [0080] In addition, as another biocompatibility test, human lysosomal extracts (derived from pooled healthy male and female livers; Sekisui Xenotech, Inc, Kansas City, KS) were found to cleave the sHBA polymer (Fig. 6). A reducing end fluorescein-tagged sHBA was treated with lysosomal enzymes (including the exoglycosidases that remove monosaccharides from the non-reducing end of GAG chains as well as sulfatases that remove sulfates from sulfated GAGs) in a low pH buffer mimicking lysosomal conditions (Xenotech, Inc) for several hours at 30°C. The sHBA polymer, as well as a positive control sulfated heparosan, was degraded as judged by the loss of signal on electrophoretic gels. This degradation indicates that unwanted accumulation and storage disorders should not be an issue for treatments with sHBA. [0081] In another set of experiments, it was also observed that myeloperoxidase (MPO), a human defense enzyme that creates ‘bleach’ in situ and is a potential biomarker for inflammation appeared to bind very well to sHBA in a set of plasma screening tests. Myeloperoxidase is a part of the host defenses, ‘sanitizing’ invaders, but when this defense component is released in excess into the blood stream, the stray MPO enzyme can bind to and damage the blood vessel walls. This MPO-based complication could add to the destruction caused by the dysregulation of complement. [0082] To confirm the plasma screening result above, recombinant human myeloperoxidase (R&D Systems, Inc; Minneapolis, MN) was found to bind to sHBA-beads in saline (0.15 M NaCl) as measured by a guaiacol MPO enzyme assay. The MPO activity could be removed from solution and then be eluted again from the beads with higher slat (>0.5 M NaCl). This indicates that sHBA, if administered in vivo, may act as a soluble ‘bait’ to help prevent myeloperoxidase from binding to HS-glycoproteins on the cell surface. This action may augment sHBA’s anti-inflammatory action on the complement system. Example 2 [0083] This example shows the structure of a sHBA embodiment analyzed by NMR. A. NMR (nuclear magnetic resonance) Methods [0084] sHBA samples were analyzed using NMR to confirm their structures. The sHBA sample was lyophilized twice from D2O (99.9% D, Aldrich) and 2.1 mg of the dry material was mixed with 1 μL 50 mM DSS-d₆ (Cambridge Isotope Laboratories) prepared in D₂O, and lyophilized again. The dry material was dissolved in 50 μL D₂O (99.96% D, Cambridge Isotope Laboratories) and transferred into a 1.7 mm NMR tube. NMR data were collected at 60°C on a Bruker Neo spectrometer (¹H, 799.71 MHz) equipped with a triple- resonance 1.7-mm cryoprobe (Bruker BioSpin, Billerica, MA). Homonuclear 2D correlation spectra (COSY, TOCSY and NOESY) were collected with 2048×200 complex points data matrix and spectral widths of 9600 and 4000 Hz (f2 and f1). TOCSY spectrum was collected with a mixing time of 70 ms and NOESY spectrum with a mixing time of 60 ms.¹H,¹³C-HSQC and HMBC spectra were collected with 200 increments and spectral widths of (¹H) 9615 and (¹³C) 14085 Hz. The HMBC spectrum was collected with a delay corresponding to a multi- bond coupling of 8 Hz. NMR data were processed in Topspin, referenced to the DSS signals (δ_H = δ_C = 0.0 ppm) and analyzed using CCPN Analysis [Vranken, W. F., Boucher, W., Stevens, T. J., Fogh, R. H., Pajon, A., Llinas,M., Ulrich, E. L., Markley, J. L., Ionides, J., and Laue, E. D. (2005) The CCPN data model for NMR spectroscopy: development of a software pipeline. Proteins 59, 687–696.]. B. Structural Results [0085] The sample of sHBA was analyzed by 1D and 2D NMR spectroscopy in order to determine the structure of the major glycan. ¹H NMR spectrum contained predominantly signals that were consistent with the presence of carbohydrates. The HSQC spectrum (Fig. 7) showed two major peaks in the anomeric region (A1 and B1), suggesting a disaccharide repeat motif. The chemical shifts of the spin systems of both residues were assigned using the combination of the 2D COSY, TOCSY, NOESY, HSQC and HMBC experiments (Table 3), and the linkage of the two residues was determined based on inter- residual correlations observed in HMBC and NOESY spectra (Fig. 7).

Table 3.¹H and ¹³C chemical shift (ppm) assignments of sugar residues of the major carbohydrate. Corresponding chemical shift differences with predicted values are shown in orange (p, pyranose ring structure; S, sulfate).

[0087] The O-sulfation of the 2-position of residue A was determined based on chemical shift differences between the experimental values and those predicted in Casper for unsubstituted polymer (Table 3), and based on mass spectrometry findings. Large down-field 1H and ¹³C chemical shift differences compared to the unsubstituted values were observed for the 2-position of residue A (2-O-sulfated Glc). The neighboring positions 1 and 3 experienced significant up-field ¹³C chemical shift differences, while the rest of the ring showed modest up-field ¹³C chemical shift differences. Additionally, the ¹³C chemical shifts of positions 4–6 in residue B were modestly affected. The overall trend of chemical shift differences is consistent with a substitution on the 2-position in residue A (Glucose of sHBA), while sulfation in particular was determined by mass spectrometry. [0088] The spectra contained additional, minor signals of sugar residues which have not been assigned by NMR due to low intensities, but LC-MS methods did find some [- 4-GlcA-1-beta-4-(6-O-sulfated Glc-1-alpha] as another sulfation site besides the more abundant 2-O-sulfate Glc residue. C. sHBA Chromatographic and Chemical Analyses: [0089] Size-exclusion chromatography light scattering [SEC-MALLS; Wyatt detectors] was used to obtain the absolute molecular weight (MW) and the polydispersity index (Mw/Mn, a measure of the size distribution of the polymer population). For example, one of the starting unsulfated HBA polymer’s MW was 27.3 kDa and the products of two different sulfation reactions, sHBA #1 or #2 (i.e. the same preparations shown on gels in Fig. 2 and tested in bioactivity assays in Table 2), were 33.8 kDa or 34.3 kDa, respectively. This calculated increase in polymer MW upon sulfation corresponds to the addition of approximately 1 sulfate per disaccharide repeating unit. Different levels of sHBA sulfation yield higher or lower polysaccharide masses, but higher than the starting HBA polymer material without any intrinsic sulfate groups. [0090] Another type of testing, combustion-based elemental analysis (Element.com; Broken Arrow, OK) measures the carbon: sulfur (C/S) ratio via infrared spectroscopy of the evolved CO₂ and SO₂ to high accuracy. In this analysis, on average approximately 1 sulfate per disaccharide was observed for the sHBA #2 preparation based on the C/S ratio of 4.56:1. For comparison, a known ‘mono-sulfated’ control, chondroitin sulfate C (from shark; BioIberica, Spain) was found to have a C/S ratio of 4.32:1, a very similar value to the sHBA #2 data. Different levels of sHBA sulfation will yield higher or lower C/S ratios for these polymers, but will always add sulfate as the starting HBA polymer is devoid of any intrinsic sulfate. . Example 3 [0091] Modeling human inflammation in animals such as a mouse is useful for predicting effects on human beings. A variety of pathogens (i.e. viruses or bacteria) or their proxies (e.g., capsid glycoproteins, lipopolysaccharide endotoxin) as well as non-infectious agents such as auto-antigens, biomaterials, or genetic mutations, trigger a hyper-inflammatory response in mice. This below experiment demonstrates that sHBA compounds were able to reduce the level of inflammation in mice resulting from a viral infection. [0092] The sHBA efficacy test used a murine-adapted influenza A virus to trigger an inflammatory response and viral infection in mice. Key symptoms and morbidity are similar in the mice and humans. This experiment assessed if sHBA administration post-infection was able to curtail inflammatory biomarkers and preserve vital organs using standard protocols and animal biosafety level 2 (ABSL2) precautions. [0093] H1N1 infection in mice. A model of virulent H1N1 Influenza A virus infection in mice was achieved by intranasal (IN) infection of the mice with an adapted A/Puerto Rico/8/1934 (PR8) virus. With a sublethal dose, infection of C57BL/6 mice with the PR8 virus resulted in acute respiratory disease and elicited innate immunity, neutralizing antibodies and cytotoxic effector and memory T cells, a profile similar to the course of infection in humans. [0094] sHBA Treatment. sHBA doses in concentration range of ~10X the ‘therapeutic anticoagulant’ range of heparin (in mice ~500 international units/kg = ~1mg/kg) were used as a treatment 2 to 4 days following infection. Note that a similar amount of heparin dose would likely cause bleeding problems. Male mice (C57BL/6; Jackson labs) were infected intranasally with a sublethal dose (~850 egg infectious units) of PR8 virus. The study entailed groups of: i. Mice infected with virus; ii. Mice treated with intranasal saline (naïve healthy control); iii. Virus-infected mice + injection of sHBA test article (0.4 mg in saline subcutaneously) in the range of 2-4 days post-infection; iv. Uninfected mice treated with sHBA 2-4 days after mock infection with saline. [0095] Mice were weighed daily to track morbidity; peak weight loss (~20%) is typically observed on Day 7-8 post-infection (p.i.). On Day 9 p.i., lungs from mice were perfused and harvested for histopathological analyses, and bronchoalveolar lavage fluid (BALF) and plasma obtained. Typically with this viral dose in C57BL/6 mice, significant numbers of inflammatory cells are present in the lungs by Day 5 p.i., and T cell responses and IFNȖ levels in BALF peak at Day 7 p.i. [0096] Monitoring cytokine/chemokine and complement post-infection and virus levels: The CCL2, CXCL10, IFNĮ, IL-1Ȗ, IL-6, IL-8, IL-12, p70, IL-10, IL-21, TNFĮ, and IFNȖ levels in the BALF may be determined by xMAP multiplex (Luminex) assays. [0097] Complement dysregulation and the cytokine storm cause much tissue and organ damage during many diseases due to the resulting hyper-inflammatory state. In Figure 8, sHBA treatment (0.4 mgs/mouse per dose, subcutaneous route between the shoulders) was shown to lower the levels of an important pro-inflammatory cytokine, IL-6, in vivo (statistics One-way ANOVA with Dunnett’s multiple comparison test; p = probability value). In this influenza A (‘flu’) challenge model with C57 black mice, the IL-6 levels (as measured by Luminex assay) in sHBA-treated C and D groups are lower in comparison to the infected mice in Group B. [0098] Shown in Fig. 8 are IL-6 cytokine levels (Luminex data) in mouse lung lavage fluid on Day 9 post-infection with influenza A strain PR8. Groups: A = uninfected mice injected with saline placebo on Days 2 and 4; B = influenza A infected mice with saline placebo on Days 2 and 4; C = infected mice with sHBA in saline on Day 2 and Day 4 post- infection; D = infected mice with sHBA in saline on Day 4 post-infection. As shown from these results, in vivo sHBA treatment led to a reduction in the cytokine storm in mice having increased inflammation due to influenza A challenge. The level of measured IL-6 was approximately 500 pg/ml in mice challenged with influenza A, but not receiving any sHBA treatment. This contrasts with mice treated with sHBA which showed a more than 50% reduction in IL-6 levels, to approximately 200 pg/ml or less. This data demonstrates that sHBA has anti-inflammatory action in vertebrates. Example 4: Treatment of a patient suffering from COVID-19 [0099] A human patient is tested and found to be positive for having a SARS-CoV- 2 viral infection. The patient is given intravenous, subcutaneous, or pulmonary (lung) administration of a composition comprising sHBA daily for 2-21 days to reduce lung inflammation in the patient. After treatment, the patient is found to have a reduced lung inflammation from the SARS-CoV-2 infection. Definitions [0100] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of ordinary skill in the art. All patents, applications, published applications and other publications referenced herein are incorporated by reference in their entirety unless stated otherwise. In the event that there are a plurality of definitions for a term herein, those in this section prevail unless stated otherwise. [0101] The term “pharmaceutically acceptable salt” refers to a salt of a compound that does not cause significant irritation to an organism to which it is administered and does not abrogate the biological activity and properties of the compound. In some embodiments, the salt is a basic addition salt of the compound. Pharmaceutical salts can be obtained by reacting a compound with inorganic acids such as hydrohalic acid (e.g., hydrochloric acid or hydrobromic acid), sulfuric acid, nitric acid and phosphoric acid. Pharmaceutical salts can also be obtained by reacting a compound with a base to form a salt such as an ammonium salt, an alkali metal salt, such as a sodium or a potassium salt, an alkaline earth metal salt, such as a calcium or a magnesium salt, a salt of organic bases such as dicyclohexylamine, N-methyl-D- glucamine, tris(hydroxymethyl)methylamine, C₁-C₇ alkylamine, cyclohexylamine, triethanolamine, ethylenediamine, and salts with amino acids such as arginine and lysine. [0102] As used herein, a “subject” refers to an animal that is the object of treatment, observation or experiment. “Animal” includes cold- and warm-blooded vertebrates and invertebrates such as fish, shellfish, reptiles and, in particular, mammals. “Mammal” includes, without limitation, mice, rats, rabbits, guinea pigs, dogs, cats, sheep, goats, cows, horses, primates, such as monkeys, chimpanzees, and apes, and, in particular, humans. In some embodiments, the subject is human. [0103] As used herein, the terms “treating,” “treatment,” “therapeutic,” or “therapy” do not necessarily mean total cure or abolition of the disease or condition. Any alleviation of any undesired signs or symptoms of a disease or condition, to any extent can be considered treatment and/or therapy. Furthermore, treatment may include acts that may worsen the patient's overall feeling of well-being or appearance. [0104] Terms and phrases used in this application, and variations thereof, especially in the appended claims, unless otherwise expressly stated, should be construed as open ended as opposed to limiting. As examples of the foregoing, the term ‘including’ should be read to mean ‘including, without limitation,’ ‘including but not limited to,’ or the like; the term ‘comprising’ as used herein is synonymous with ‘including,’ ‘containing,’ or ‘characterized by,’ and is inclusive or open-ended and does not exclude additional, unrecited elements or method steps; the term ‘having’ should be interpreted as ‘having at least;’ the term ‘includes’ should be interpreted as ‘includes but is not limited to;’ the term ‘example’ is used to provide exemplary instances of the item in discussion, not an exhaustive or limiting list thereof; and use of terms like ‘preferably,’ ‘preferred,’ ‘desired,’ or ‘desirable,’ and words of similar meaning should not be understood as implying that certain features are critical, essential, or even important to the structure or function, but instead as merely intended to highlight alternative or additional features that may or may not be utilized in a particular embodiment. In addition, the term “comprising” is to be interpreted synonymously with the phrases "having at least" or "including at least". When used in the context of a process, the term "comprising" means that the process includes at least the recited steps, but may include additional steps. When used in the context of a compound, composition or device, the term "comprising" means that the compound, composition or device includes at least the recited features or components, but may also include additional features or components. Likewise, a group of items linked with the conjunction ‘and’ should not be read as requiring that each and every one of those items be present in the grouping, but rather should be read as ‘and/or’ unless expressly stated otherwise. Similarly, a group of items linked with the conjunction ‘or’ should not be read as requiring mutual exclusivity among that group, but rather should be read as ‘and/or’ unless expressly stated otherwise. [0105] With respect to the use of substantially any plural and/or singular terms herein, those having skill in the art can translate from the plural to the singular and/or from the singular to the plural as is appropriate to the context and/or application. The various singular/plural permutations may be expressly set forth herein for sake of clarity. The indefinite article “a” or “an” does not exclude a plurality. A single processor or other unit may fulfill the functions of several items recited in the claims. The mere fact that certain measures are recited in mutually different dependent claims does not indicate that a combination of these measures cannot be used to advantage. Any reference signs in the claims should not be construed as limiting the scope. [0106] It is understood that the methods and combinations described herein include crystalline forms (also known as polymorphs, which include the different crystal packing arrangements of the same elemental composition of a compound), amorphous phases, salts, solvates and hydrates. In some embodiments, the compounds described herein exist in solvated forms with pharmaceutically acceptable solvents such as water, ethanol, or the like. In other embodiments, the compounds described herein exist in unsolvated form. Solvates contain either stoichiometric or non-stoichiometric amounts of a solvent, and may be formed during the process of crystallization with pharmaceutically acceptable solvents such as water, ethanol, or the like. Hydrates are formed when the solvent is water, or alcoholates are formed when the solvent is alcohol. In addition, the compounds provided herein can exist in unsolvated as well as solvated forms. In general, the solvated forms are considered equivalent to the unsolvated forms for the purposes of the compounds and methods provided herein. [0107] Where a range of values is provided, it is understood that the upper and lower limit, and each intervening value between the upper and lower limit of the range is encompassed within the embodiments. [0108] The term “analog” as used herein will be understood to refer to a variation of the normal or standard form or the wild-type form of molecules. For polypeptides or polynucleotides, an analog may be a variant (polymorphism), a mutant, and/or a naturally or artificially chemically modified version of the wild-type polynucleotide (including combinations of the above). For oligosaccharides and polysaccharides, an analog may be a variant structure or artificially chemically or enzymatically modified version of the wild-type or the original carbohydrate (including combinations of the above). Such analogs may have higher, full, intermediate, or lower activity than the normal form of the molecule, or no activity at all; in the latter case, these drugs can often act as bait or blockers of activity. Alternatively and/or in addition thereto, for a chemical, an analog may be any structure that has the functionalities (including alterations or substitutions in the core moiety) desired, even if comprised of different atoms or isomeric arrangements. [0109] The term "pharmaceutically acceptable" refers to compounds and compositions that are suitable for administration to humans and/or animals without undue adverse side effects such as toxicity, irritation and/or allergic response commensurate with a reasonable benefit/risk ratio. [0110] Certain abbreviations used within the context of this disclosure include, but are not limited to: Glc, glucose; GlcA, glucuronic acid; GlcNAc, N-acetylglucosamine;; PmHS, P. multocida HEP synthase; UDP, uridine diphosphate; HBA, hepbiuronic acid; sHBA, sulfated or sulfonated hepbiuronic acid; MW, molecular weight; GAG, glycosaminoglycan. [0111] Pharmaceutical compositions [0112] Some embodiments described herein relates to a pharmaceutical composition, which can include an effective amount of sHBA, or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier, diluent, excipient or combination thereof. [0113] The term “pharmaceutical composition” refers to a mixture of sHBA with other chemical components, such as diluents or carriers. The pharmaceutical composition facilitates administration of the compound to an organism. Pharmaceutical salts can be obtained by reacting a compound with a base to form a salt such as an ammonium salt, an alkali metal salt, such as a sodium or a potassium salt, an alkaline earth metal salt, such as a calcium or a magnesium salt, a salt of organic bases such as dicyclohexylamine, N-methyl-D- glucamine, tris(hydroxymethyl)methylamine, C₁-C₇ alkylamine, cyclohexylamine, triethanolamine, ethylenediamine, and salts with amino acids such as arginine and lysine. Pharmaceutical compositions can also be obtained by reacting compounds with inorganic or organic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid and salicylic acid. Pharmaceutical compositions will generally be tailored to the specific intended route of administration. A pharmaceutical composition is suitable for human and/or veterinary applications. [0114] As used herein, a “carrier” refers to a compound that facilitates the incorporation of a compound into cells or tissues. For example, without limitation, dimethyl sulfoxide (DMSO), Ethanol (EtOH), or PEG400 is a commonly utilized carrier that facilitates the uptake of many organic compounds into cells or tissues of a subject. [0115] As used herein, a “diluent” refers to an ingredient in a pharmaceutical composition that lacks pharmacological activity but may be pharmaceutically necessary or desirable. For example, a diluent may be used to increase the bulk of a potent drug whose mass is too small for manufacture and/or administration. It may also be a liquid for the dissolution of a drug to be administered by injection, ingestion or inhalation. A common form of diluent in the art is a buffered aqueous solution such as, without limitation, phosphate buffered saline that mimics the composition of human blood. [0116] As used herein, an “excipient” refers to an inert substance that is added to a pharmaceutical composition to provide, without limitation, bulk, consistency, stability, binding ability, lubrication, disintegrating ability etc., to the composition. A “diluent” is a type of excipient. [0117] The pharmaceutical compositions described herein can be administered to a human patient per se, or in pharmaceutical compositions where they are mixed with other active ingredients, as in combination therapy, or carriers, diluents, excipients or combinations thereof. Proper formulation is dependent upon the route of administration chosen. Techniques for formulation and administration of the compounds described herein are known to those skilled in the art. [0118] The pharmaceutical compositions disclosed herein may be manufactured in a manner that is itself known, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or tableting processes. Additionally, the active ingredients are contained in an amount effective to achieve its intended purpose. Many of the compounds used in the pharmaceutical combinations disclosed herein may be provided as salts with pharmaceutically compatible counterions. [0119] Multiple techniques of administering a compound exist in the art including, but not limited to, oral, rectal, topical, aerosol, injection and parenteral delivery, including intramuscular, subcutaneous, intravenous, intramedullary injections, intrathecal, direct intraventricular, intraperitoneal, intranasal and intraocular injections. [0120] One may also administer the compound in a local rather than systemic manner, for example, via injection of the compound directly into the infected area, often in a depot or sustained release formulation. Furthermore, one may administer the compound in a targeted drug delivery system, for example, in a liposome coated with a tissue-specific antibody. The liposomes will be targeted to and taken up selectively by the organ. [0121] The pharmaceutical compositions may, if desired, be presented in a pack or dispenser device that may contain one or more unit dosage forms containing the active ingredient. The pack may for example comprise metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by instructions for administration. The pack or dispenser may also be accompanied with a notice associated with the container in form prescribed by a governmental agency regulating the manufacture, use, or sale of pharmaceuticals, which notice is reflective of approval by the agency of the form of the drug for human or veterinary administration. Such notice, for example, may be the labeling approved by the U.S. Food and Drug Administration for prescription drugs, or the approved product insert. Compositions that can include sHBA formulated in a compatible pharmaceutical carrier may also be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition. [0122] The above description discloses several methods and materials of the present invention. This invention is susceptible to modifications in the methods and materials, as well as alterations in the fabrication methods and equipment. Such modifications will become apparent to those skilled in the art from a consideration of this disclosure or practice of the invention disclosed herein. Consequently, it is not intended that this invention be limited to the specific embodiments disclosed herein, but that it cover all modifications and alternatives coming within the true scope and spirit of the invention. [0123] All references cited herein, including but not limited to published and unpublished applications, patents, and literature references, are incorporated herein by reference in their entirety and are hereby made a part of this specification. To the extent publications and patents or patent applications incorporated by reference contradict the disclosure contained in the specification, the specification is intended to supersede and/or take precedence over any such contradictory material.

Claims

WHAT IS CLAIMED IS: 1. A pharmaceutical composition comprising at least one sulfated hepbiuronic acid (sHBA) repeating disaccharide unit. 2. The pharmaceutical composition of claim 1, wherein the number of sulfated hepbiuronic acid (sHBA) repeating disaccharide units in each polymer is from 2 to 1,000. 3. The pharmaceutical composition of claims 1 or 2, further comprising a pharmaceutically acceptable carrier or diluent. 4. The pharmaceutical composition of any of claims 1-3, wherein the sHBA comprises units of 2-O-sulfated-Glucose. 5. The pharmaceutical composition of any of claims 1-4, wherein the sHBA comprises units of 6-O-sulfated-Glucose. 6. The pharmaceutical composition of any of claims 1-5, wherein the Glucose in the sHBA is less than 100% sulfated. 7. The pharmaceutical composition of any of claims 1-5, wherein the Glucose in the sHBA is less than 90% sulfated. 8. The pharmaceutical composition of any of claims 1-5, wherein the Glucose in the sHBA is less than 80% sulfated. 9. A method of reducing inflammation in a subject, comprising: determining a subject in need of treatment for inflammation; and administering to the subject a therapeutically effective amount of the pharmaceutical composition of claims 1-8, wherein the administration results in reduced inflammation in the subject. 10. The method of claim 9, wherein the inflammation is inflammation derived from a microbial (viral or bacterial) infection. 11. The method of claim 9, further comprising administering another therapeutic agent to said subject. 12. The method of claim 11, wherein the pharmaceutical composition and the other therapeutic agent are co-administered. 13. The method of claim 11, wherein the other therapeutic agent is an interferon, a SARS-CoV-2 RNA-dependent RNA polymerase inhibitor, a cap-dependent endonuclease, a protease inhibitor, nucleotide analog, therapeutic antibody or conjugate, antibiotic, or a spike inhibitor. 14. The method of claim 11, wherein both the pharmaceutical composition and the other therapeutic agent are dispersed or dissolved together in a pharmaceutically-acceptable carrier. 15. The method of claim 9, wherein the administration is intravenous or subcutaneous or intraperitoneal administration. 16. The method of claim 9, wherein the administration is pulmonary or nebulized- based administration. 17. The method of claim 16, wherein the administration is of a solution comprising the pharmaceutical composition or pharmaceutically acceptable salt in a concentration between 0.1 mg/ml and 500 mg/ml. 18. The method of claim 9, wherein the pharmaceutical composition or pharmaceutically acceptable salt thereof administered to the subject is in an amount from about 0.1 mg to about 1,000 mg of the pharmaceutical composition per kg of body weight of the subject.